Refolded recombinant beta-secretase crystals and methods for preparing and using the same

ABSTRACT

The present application relates to methods for growing crystals of both the uncomplexed and complexed forms of β-secretase (BACE) polypeptide. The present application also relates to crystalline forms of uncomplexed BACE and the three-dimensional structure of BACE, as determined from the crystals. In addition, the present application relates to the use of crystalline forms of BACE to identify ligands, preferably inhibitors (antagonists), which bind to, and preferably inhibit the enzymatic activity of, BACE. Furthermore, the present application relates to nucleic acid sequences encoding BACE polypeptide, and methods for making BACE in greater quantity than prior methods, resulting in more effective crystallization.

This application is a division of copending U.S. patent application Ser.No. 10/443949, filed May 22, 2003, which claims the benefit of U.S.provisional patent application No. 60/383,480, filed May 24, 2002, eachof which is herein incorporated by reference in its entiretry.

BACKGROUND OF THE INVENTION

All publications cited in the present application are incorporated intheir entirety by reference.

1. Field of the Invention

The present application relates to nucleic acids encoding β-secretase(BACE) polypeptides, methods for making BACE polypeptides, methods forgrowing crystals of BACE, crystalline BACE, the three-dimensionalstructure of BACE, and the use of the crystalline forms to identifyligands, such as antagonists, that bind to BACE.

2. Invention Background

Alzheimer's disease (AD) is a neurodegenerative disease characterized byneuronal loss due to the extracellular accumulation of amyloid plaquesand intracellular accumulation of neurofibrillary tangles in the brain(reviewed by Selkoe, D.J. (1999) Nature 399: A23-31). Two majorcomponents of amyloid plaques are small peptide fragments Aβ40 and Aβ42,which are generated from cleavage of the membrane-anchored amyloidprecursor protein (APP) by the proteolytic activity of β- andγ-secretases. APP is a type I integral membrane protein containing theAβ segment, which begins at D672 in the longest isoform and spans theboundary of the exocytoplasmic region (28 amino acids) and thetransmembrane domain (12-14 amino acids). The γ-secretase activitycleaves APP within the transmembrane domain to produce thecarboxy-terminal end of Aβ polypeptide. The β-secretase activity(aspartic protease activity), identified in a protein that is known as“mamapsin 2”, “human β-site APP-cleaving enzyme” or “BACE”, and “Asp 2”,cleaves APP on the extracellular side of the membrane to produce theamino-terminal end of Aβ. (Vassar, R. et al., (1999) Science 286,735,Sinha, S. et al., (1999) Nature 402,537, Yan, R. et al., (1999) Nature402,522, Hussain, I. et al., (1999) Mol. Cell Neurosci. 14, 419 and Lin,X. (2000) et al., Proc. Natl. Acad. Sci. USA 97,1456. Another enzyme,known as α-secretase, cleaves APP at a position within the Aβ sequenceto produce a soluble APPα (Esch et al., (1990) Science 248: 1122-1124).

During the course of AD, Aβ polypeptide accumulates extracellularly inthe brain, and forms large, insoluble amyloid fibrils that elicit bothcytotoxic and inflammatory responses. Thus, BACE and γ-secretaseproteases are targets for potential inhibitor drugs (antagonists)against AD. Because it was discovered that BACE activity is therate-limiting step in Aβ production in vivo (Sinha and Lieberburg,(1999) Proc. Natl. Acad. Sci. USA 96: 11049), BACE has become a primetarget for the development of inhibitors (antagonists) to treat AD.

The BACE gene encodes a 501 residue polypeptide having, from N- toC-terminus, an N-terminal signal sequence of 21 amino acids; apro-protein domain of 22 to 45 residues, which is proteolyticallyremoved by furin to generate mature β-secretase (Creemers, J.W., et al.(2001) J. Biol. Chem. 276: 4211-4217; Bennet, B.D., et al. (2000) J.Biol. Chem. 275: 37712-37717); a protease (catalytic) domain; aconnecting strand, an integral membrane (transmembrane) domain of about17 amino acids; and a short cytosolic C-terminal tail of 24 amino acids(Vassar et al., supra). Sequence analyses indicate that BACE belongs toa subfamily of membrane-bound and soluble proteases, and contains aclassic consensus active site motif found in aspartyl proteases (D T/S GT/S) at positions 93 to 96 and 289 to 292. The entire BACE sequencedisplays only mild homology with known aspartyl proteases, approximately30% identity and 37% similarity with members of the mammalian pepsinfamily, with the highest homology found in the central portion of theextracellular domain.

Accurate information regarding the three-dimensional structure ofβ-secretase is helpful in the design and identification of ligands,particularly inhibitors (antagonists), of BACE, and in the enzymaticcharacterization of the enzyme. This information may be provided usingcrystals of the protein in X-ray crytallographic analysis.

Crystallization of a protein is a very time consuming and complexprocess. Crystallization of a protein requires a precise set of reagentsand reaction conditions that promote the growth of crystallized protein.For example, specific amounts of protein, buffer, precipitating agentand salt, at a precise temperature, are required to produce X-raydiffraction quality crystals. There are an infinite number ofcombinations of the above reagents and reaction conditions. Therefore,the number of different combinations that can be tested is limited bythe amount of protein that can be produced. Because the precise set ofconditions that will produce crystals can not be predicted, one is morelikely to discover crystals as more reagents and reaction conditions aretested. As a result, effective crystallization requires a large amountof refolded protein, typically milligram quantities. This is problematicbecause current methods for expressing BACE in E. coli provide lowyields of unfolded protein. In addition, large amounts of unfoldedprotein are required to optimize the protein's refolding procedures.Thus, there is a need for nucleic acids encoding BACE that are optimizedfor E. coli expression, which utilize codons that are preferred by E.coli, to produce large quantities of BACE to both discover optimalrefolding conditions and so that many different combinations of theabove reagents and reaction conditions may be tested in order tooptimize the crystallization conditions for BACE.

A crystal form of β-secretase complexed to an inhibitor is described inHong et al., (2000) Science 290:150-153. In addition, severalinternational applications published under the Patent CooperationTreaty, international publication numbers WO 02/25276 A1, WO 01/00663 A2and WO 01/00665 A2, provide crystal forms of BACE complexed to aninhibitor. Knowledge of the structure of a protein in both theuncomplexed and complexed forms allows one to determine how thethree-dimensional structure of the protein changes upon binding to aligand. This aids in structure based drug design because it providesmore information regarding how a particular ligand may be altered toincrease its binding to the protein. Thus, there is a need for crystalsof β-secretase which have similar structure and activity to that ofnative BACE, and which can be produced in the uncomplexed form.

SUMMARY OF THE INVENTION

The present invention addresses the foregoing needs by providing nucleicacids encoding BACE polypeptides that are able to produce largequantities of BACE when expressed in E. coli cells. The presentinvention also addresses the foregoing needs by providing crystals ofBACE in the uncomplexed form.

An embodiment of the invention provides an isolated or recombinantnucleic acid comprising the nucleotide sequence set forth in SEQ IDNO: 1. A further embodiment provides a nucleic acid comprising thenucleotide sequence set forth in SEQ ID NO: 17. An additional embodimentof the invention provides an expression vector comprising a nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO: 17. A furtherembodiment of the invention provides a host cell comprising the abovevector.

An embodiment of the invention also provides a method for makingβ-secretase polypeptide comprising transforming a host cell with anexpression vector comprising an isolated or recombinant nucleic acidcomprising the nucleotide sequence set forth in SEQ ID NO: 17 underconditions in which the polypeptide is expressed. Preferably, the hostcell is a bacterial cell. More preferably, the bacterial cell is an E.coli cell. Most preferably, the E. coli cell is a BL21 (DE3)Star cell.In addition, the vector is preferably pET 11a. Preferably, the methodfurther comprises a refolding step wherein the polypeptide is refoldedin the presence of about 0.5 mM reduced glutathione and about 0.5 mMoxidized glutathione.

In addition, the method for making β-secretase polypeptide furthercomprises a processing step wherein the polypeptide is exchanged intoabout 20 mM Hepes at about pH 7.5 and about 150 mM NaCl and thenconcentrated to about 5 mg/ml, and incubated at about 4° C. for abouttwo weeks to form a processed polypeptide. Preferably, the processedpolypeptide comprises the amino acid sequence set forth in either SEQ IDNO: 20 or SEQ ID NO: 22. Alternatively, the method for makingβ-secretase polypeptide further comprises a processing step wherein thepolypeptide is exchanged into about 20 mM Hepes at about pH 7.5 andabout 150 mM NaCl and then concentrated to about 15 mg/ml, and incubatedat about room temperature for about 72 hours to form a processedpolypeptide. Preferably, the processed polypeptide comprises the aminoacid sequence set forth in either SEQ ID NO: 20 or SEQ ID NO: 22.

An embodiment of the invention provides a method for growing a crystalcomprising adding about 16 mg/ml of a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 22 to a crystallization solution,the solution comprising about 13.75% to about 15.0% PEG3350 and about0.6 M ammonium iodide, and crystallizing the solution at about 4° C.using a hanging drop method.

Another embodiment of the invention provides a method for growing acrystal comprising a polypeptide complexed to a ligand comprising addingabout 16 mg/ml of a polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 22 and about 0.5 mM to about 1.0 mM of the ligand toa crystallization solution, the solution comprising about 20% PEG3350and about 0.2 M ammonium tartrate, and crystallizing the solution atabout 4° C. using a hanging drop method. Preferably, the ligand is anantagonist.

An embodiment of the invention provides a crystal that is made by addingabout 16 mg/ml of a polypeptide comprising the amino acid sequence setforth in SEQ 5 ID NO: 22 to a crystallization solution, the solutioncomprising about 13.75% to about 15.0% PEG3350 and about 0.6 M ammoniumiodide, and crystallizing the solution at about 4° C. using a hangingdrop method. Preferably, the crystal has a space group of C2 with unitcell dimensions of a=236.0 Å, b=103.6 Å and c=65.0 Å. Alternatively, thecrystal has a space group of C2 with unit cell dimensions wherein aranges from about 231.3 Å to about 240.7 Å, b ranges from about 101.5 Åto about 105.7 Å, and c ranges from about 63.7 Å to about 66.3 Å. Thecrystal is preferably characterized by the structure coordinates setforth in Table 1. Preferably, the crystal effectively diffracts X-raysfor determination of atomic coordinates of the polypeptide to aresolution of greater than about 5.0 Å.

Another embodiment of the invention provides a crystal of an uncomplexedβ-secretase polypeptide wherein the β-secretase polypeptide is expressedin E. coli cells comprising a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 22, wherein the crystal effectivelydiffracts X-rays for determination of atomic coordinates of thepolypeptide to a resolution of greater than about 5.0 Å. Preferably, 20the crystal has a space group of C2 with unit cell dimensions of a=236.0Å, b=103.6 Å and c=65.0 Å. Alternatively, the crystal has a space groupof C2 with unit cell dimensions wherein a ranges from about 231.3 Å toabout 240.7 Å, b ranges from about 101.5 Å to about 105.7 Å, and cranges from about 63.7 Å to about 66.3 Å.

An embodiment of the invention provides a crystal of an uncomplexedβ-secretase polypeptide characterized by the structure coordinates setforth in Table 1. A further embodiment of the invention provides acrystal of an uncomplexed β-secretase polypeptide characterized bystructure coordinates comprising a root mean square deviation ofconserved residue backbone atoms of less than about 1.5 Å whensuperimposed on backbone atoms described by the structure coordinatesset forth in Table 1. Preferably, the root mean square deviation is lessthan about 1.0 Å. More preferably, the root mean square deviation isless than about 0.5 Å. Most preferably, the root mean square deviationis less than about 0.1 Å. Preferably, the crystal effectively diffractsX-rays for determination of atomic coordinates of the polypeptide to aresolution of greater than about 5.0 Å. The crystal preferably has aspace group of C2 with unit cell dimensions of a=236.0 Å, b=103.6 Å andc=65.0 Å. Alternatively, the crystal has a space group of C2 with unitcell dimensions wherein a ranges from about 231.3 Å to about 240.7 Å, branges from about 101.5 Å to about 105.7 Å, and c ranges from about 63.7Å to about 66.3 Å.

An embodiment of the invention provides a magnetic data storage mediumcomprising the structure coordinates set forth in Table 1. Anotherembodiment of the invention provides a computer for producing athree-dimensional representation of β-secretase polypeptide which isdefined by the structure coordinates set forth in Table 1, or athree-dimensional representation of a homologue of the β-secretaseprotein wherein the homologue has a root mean square deviation from thebackbone atoms set forth in Table 1 of less than about 1.5 Å, whereinthe computer comprises: (a) a machine-readable data storage mediumcomprising a data storage material encoded with machine-readable data,wherein the data comprises the structure coordinates set forth in Table1; (b) a working memory for storing instructions for processing themachine-readable data; (c) a central-processing unit coupled to theworking memory and to the machine-readable data storage medium forprocessing the machine readable data into the three-dimensionalrepresentation; and (d) a display coupled to the central-processing unitfor displaying the three-dimensional representation. Preferably, theroot mean square deviation is less than about 1 Å. More preferably, theroot mean square deviation is less than about 0.5 Å. Most preferably,the root mean square deviation is less than about 0.1 Å.

An embodiment of the invention provides a method for identifying aligand that binds to β-secretase comprising: (a) obtaining a set ofatomic coordinates defining the three-dimensional structure of a crystalof an uncomplexed, processed β-secretase polypeptide expressed in E.coli cells that effectively diffracts X-rays for determination of theatomic coordinates of the β-secretase polypeptide to a resolution ofgreater than about 5.0 Å; (b) selecting a ligand by performing rationaldrug design with the set of atomic coordinates obtained in step (a),wherein the selecting is performed in conjunction with computermodeling; (c) contacting the ligand with the polypeptide; and (d)detecting binding of the ligand to the polypeptide. Preferably, themethod provides a crystal having a space group of C2 with unit celldimensions of a=236.0 Å, b=103.6 Å and c=65.0 Å. Alternatively, themethod provides a crystal having a space group of C2 with unit celldimensions wherein a ranges from about 231.3 Å to about 240.7 Å, branges from about 101.5 Å to about 105.7 Å, and c ranges from about 63.7Å to about 66.3 Å.

An embodiment of the invention provides a method for identifying aligand that binds to β-secretase comprising: (a) preparing a mixture ofβ-secretase with a potential ligand comprising adding about 1.5 to about5 molar ratio of ligand to about 16 mg/ml of β-secretase comprising theamino acid sequence set forth in SEQ ID NO: 22; (b) crystallizing themixture to form a crystal; and (c) performing X-ray diffraction analysison the crystal.

Another embodiment of the invention provides a method for identifying aligand that binds to β-secretase comprising: (a) soaking a crystal,which is made by adding about 16 mg/ml of a polypeptide comprising theamino acid sequence set forth in SEQ ID NO: 22 to a crystallizationsolution, the solution comprising about 13.75% to about 15% PEG3350 andabout 0.6 M ammonium iodide, and crystallizing the solution at about 4°C. using a hanging drop method, in a solution comprising the ligand; and(b) performing X-ray diffraction on the crystal.

An additional embodiment of the invention provides a method foridentifying a ligand that binds to β-secretase comprising: (a) preparinga mixture of β-secretase with a ligand comprising adding a first ligandto about 16 mg/ml of β-secretase comprising the amino acid sequence setforth in SEQ ID NO: 22; (b) crystallizing the mixture to form a crystal;(c) soaking the crystal in a solution comprising a potential ligand,wherein the potential ligand displaces the first ligand from thecrystal; and (d) performing X-ray diffraction on the crystal.

An embodiment of the invention provides a method for identifying aβ-secretase antagonist comprising the steps of: (a) selecting apotential antagonist by performing rational drug design using thethree-dimensional structure of a crystal of an uncomplexed β-secretasewherein the β-secretase polypeptide is expressed in E, coli cells andcomprises the amino acid sequence set forth in SEQ ID NO: 22, whereinthe crystal effectively diffracts X-rays for determination of atomiccoordinates of the polypeptide to a resolution of greater than about 5.0Å, and wherein the selecting is performed in conjunction with computermodeling; (b) contacting the potential antagonist with β-secretase; and(c) detecting binding of the potential antagonist to the β-secretase,wherein an antagonist is identified on the basis of its ability toinhibit the catalytic activity of the β-secretase.

An embodiment of the invention provides a method for identifying aninhibitor of β-secretase comprising: (a) obtaining a set of atomiccoordinates from a crystal defining the three-dimensional structure ofan uncomplexed, processed β-secretase polypeptide expressed in E. colicells; (b) selecting a potential inhibitor by performing rational drugdesign with the set of atomic coordinates obtained in step (a), whereinthe selecting is performed in conjunction with computer modeling; (c)contacting the potential inhibitor with a β-secretase protein; and (d)measuring the activity of the protein, wherein the potential inhibitoris identified when there is a decrease in activity of the β-secretase inthe presence of the inhibitor as compared to the activity of β-secretasein the absence of the potential inhibitor. Preferably, the methodprovides a crystal having a space group of C2 with unit cell dimensionsof a=236.0 Å, b=103.6 Å and c=65.0 Å. Alternatively, the method providesa crystal having a space group of C2 with unit cell dimensions wherein aranges from about 231.3 Å to about 240.7 Å, b ranges from about 101.5 Åto about 105.7 Å, and c ranges from about 63.7 Å to about 66.3 Å.

An embodiment of the invention provides a method for identifying apotential inhibitor of β-secretase comprising the steps of: (a) viewinga three-dimensional structure of the β-secretase as defined by theatomic coordinates of β-secretase set forth in Table 1; (b) employingthe three-dimensional structure to design or select the potentialinhibitor; (c) synthesizing the potential inhibitor; and (d) contactingthe potential inhibitor with the β-secretase in the presence of asubstrate to determine the ability of the potential inhibitor to inhibitthe β-secretase.

DETAILED DESCRIPTION OF THE INVENTION

The present application relates to nucleic acids encoding β-secretase(BACE) polypeptides; methods for making, refolding and processing BACEpolypeptides; methods for growing crystals of BACE in both theuncomplexed and complexed forms; crystalline BACE; the three-dimensionalstructure of BACE, and the use of the crystalline forms to identifyligands, such as antagonists, that bind to BACE.

Methods for Producing β-secretase Nucleic Acids and/or Polypeptides

Embodiments of the invention provide methods for producing β-secretase(BACE) nucleic acids and/or polypeptides. A BACE nucleic acid orpolypeptide can be produced by any conventional method, including, butnot limited to, synthetic methods, such as solid phase, liquid phase,and combination solid/liquid phase polypeptide syntheses; recombinantDNA methods, including cDNA cloning, optionally combined withsite-directed mutagenesis; and/or purification of the natural products,optionally combined with enzymatic or chemical cleavage methods toproduce fragments of naturally-occurring BACE nucleic acids orpolypeptides.

In addition, a BACE nucleic acid or polypeptide can be any form of BACEfrom any species. Preferably, the BACE nucleic acid or polypeptide isfrom an animal. More preferably, the BACE nucleic acid or polypeptide isfrom a mammal, including, but not limited to, mouse, rat, rabbit, dog,or human. Most preferably, the BACE nucleic acid or polypeptide is froma human.

Preferably, the BACE polypeptide is structurally and functionallysimilar to naturally-occurring human BACE. However, the BACE polypeptideneed not be glycosylated or include any sort of post-translationalmodification.

Preferably, a BACE polypeptide is produced from a synthetic BACE genethat contains an optimized spelling of the native nucleotides of thefirst approximately one-third of the human BACE gene. The spelling ofthe nucleotide sequence is optimized by increasing or decreasing the GCcontent of the sequence to approximately 50% and by optimizing the codonusage for a particular expression system, yet keeping the resultingamino acid sequence unchanged from the native sequence. Decreasing theGC content of the nucleotide sequence reduces the potential forsecondary structure formation of mRNA, which results in decreased levelsof protein expression. The codon usage was optimized by using codonsthat are preferred in E. Coli. Preferred codons are determined bysequencing genomic DNA of the host organism and applying statisticalanalysis to determine which codons are preferred in nature. Preferably,the synthetic optimized BACE gene used to produce the BACE polypeptidecomprises the nucleotide sequence set forth in SEQ ID NO: 17. Mostpreferably, the synthetic optimized BACE gene used to produce the BACEpolypeptide comprises the nucleotide sequence set forth in SEQ ID NO: 1.Alternatively, a BACE polypeptide can be produced comprising the aminoacid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 18.

A BACE gene comprising the nucleotide sequence set forth in either SEQID NO: 1 or SEQ ID NO: 17, or a BACE polypeptide comprising the aminoacid sequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 18 can beproduced by any conventional molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed.1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds.(1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds.(1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; ImmobilizedCells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide ToMolecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, Inc. (1996) (herein “Ausubel etal., 1996”).

Most preferably, the nucleotide sequence set forth in SEQ ID NO: 1 isgenerated using polymerase chain reaction (PCR), as described in example1 below. Briefly, and described in more detail in example 1, a threestage PCR strategy is adopted to construct a soluble synthetic BACEgene. In the first stage, primers are generated which are used toamplify two half fragments. In the second stage, the two half fragmentsare used to amplify the synthetic fragment of 1-420 bp of BACE (SEQ IDNO: 1). Finally, in the third stage, the synthetic soluble BACEcomprising nucleotides 1-1362 (SEQ ID NO: 17) is amplified.

In addition to being derived from the above optimized nucleic acid, theBACE gene preferably includes an additional nucleotide sequence to driveprotein expression, such as a T7 tag located on the amino terminus. TheT7 tag preferably comprises a nucleotide sequence encoding the aminoacid sequence MASMTGGQQMG (SEQ ID NO: 14). The BACE gene also preferablyincludes a C-terminal truncation that omits the transmembrane domain ofthe native BACE enzyme, which aids in purification of the protein. TheC-terminal truncation preferably omits the nucleotide sequence encodingthe final approximately 40-60 amino acid residues of the protein. Morepreferably, the C-terminal truncation omits the nucleotide sequenceencoding the final 47 amino acid residues of the polypeptide so that thepolypeptide is truncated after amino acid residue 454 of the nativeenzyme. However, the nucleotide or amino acid sequence need not includeany N-terminal or C-terminal additions and/or truncations.

Finally, the synthetic BACE gene may be expressed. The terms “express”and “expression” mean allowing or causing the information in a gene ornucleotide sequence to become manifest, e.g., producing a protein byactivating the cellular functions involved in transcription and,optionally, translation of a corresponding gene or nucleotide sequence.A nucleotide sequence can be expressed using a vector, such as pET 11a.Alternatively, a nucleotide sequence can be expressed using in vitrotranslation systems (e.g., rabbit reticulocyte lysate-based systems) orin or by a cell to form an “expression product” such as an mRNA or aprotein. The expression product, e.g., the resulting protein, may alsobe referred to as “expressed”. The BACE polypeptide may be expressed inany type of host cell. Preferably, the polypeptide is expressed inmammalian cells, insect cells or bacterial cells. More preferably, thepolypeptide is expressed in E. coli cells. Most preferably, thepolypeptide is expressed in BL21 (DE3)Star cells. Therefore, theresulting polypeptide is not post-translationally modified. However, thepresent invention contemplates crystals comprising BACE polypeptidewhich have been modified (e.g., post-translationally modified) in anymanner, such as glycosylation, phosphorylation, sulfonation, orPEGylation. The optimized nucleotide sequences set forth in both SEQ IDNO: 1 and SEQ ID NO: 17 unexpectedly result in an increase in insolubleprotein expression of approximately four fold when expressed in BL21(DE3)Star cells.

An embodiment of the present invention provides polypeptides that differfrom the BACE polypeptide comprising the amino acid sequence set forthin either SEQ ID NO: 2 or SEQ ID NO: 18 by having amino acid deletions,substitutions, and additions. Preferably, the BACE polypeptide used inthe present invention contains catalytic (proteolytic) properties thatare comparable to those that have been reported for synthetic peptidesderived from the β-amyloid precursor protein (APP) peptide sequence.Examples of APP peptides which may be cleaved by BACE of the presentinvention are disclosed, for example, in Lin et al., (2000) Proc. Nat.Acad. Sci., 97(4):1456-1460 and Turner et al., (2001) Biochemistry,40(34):10,001-10,006. The bilobal protein, typically, is lightlyglycosylated with glycan attachment, accounting for approximately 4 kDof the protein's molecular weight.

An embodiment of the present invention also provides various mutantforms, homologues and variants of BACE. The terms “mutant” and“mutation” mean any detectable change in genetic material, e.g., DNA, orany process, mechanism, or result of such a change. Therefore,embodiments of the invention provide nucleic acids which differ from thenucleotide sequence set forth in either SEQ ID NO: 1 or SEQ ID NO: 17.This includes gene mutations in which the structure (e.g., DNA sequence)of a gene is altered, any gene or DNA arising from any mutation process,and any expression product (e.g., protein) expressed by a modified geneor DNA sequence. The term “variant” may also be used to indicate amodified or altered gene, DNA sequence, polypeptide or enzyme, etc.,i.e., any kind of mutant. Sequence- and function-conservative variantsof BACE polypeptides are also contemplated for use in the presentinvention. “Sequence-conservative variants” of BACE are those in which achange of one or more nucleotides in a given codon position results inno alteration in the amino acid encoded at that position.“Function-conservative variants” of BACE are those in which a givenamino acid residue in a BACE polypeptide has been changed withoutaltering the overall conformation and function of the polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar properties, such as, for example, polarity, hydrogenbonding potential, acidic, basic, hydrophobic, aromatic, and the like.

Protein or polypeptide homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See, e.g., Needleham, et al. J. Mol. Biol. 48:443-453 (1970);Sankoff et al., “Time Warps, String Edits, and Macromolecules: TheTheory and Practice of Sequence Comparison”, Ch. 1, Addison-Wesley,Reading, Mass. (1983); and software packages from IntelliGenetics,Mountain View, Calif. and the University of Wisconsin Genetics ComputerGroup (GCG), Madison, Wis. This changes when considering conservativesubstitutions as matches. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylaianine,tyrosine. Homologous amino acid sequences are intended to includenatural variations of the BACE amino acid sequence. Typical homologousBACE polypeptides used in this invention will have from 50-100% homology(if gaps can be introduced), to 60-100% homology (if conservativesubstitutions are included), e.g., with BACE comprising the amino acidsequence set forth in either SEQ ID NO: 2 or SEQ ID NO: 18. Homologymeasures are preferably at least about 70%, generally at least 76%, moregenerally at least 81 %, often at least 85%, more often at least 88%,typically at least 90%, more typically at least 92%, usually at least94%, more usually at least 95%, preferably at least 96%, and morepreferably at least 97%, and in particularly preferred embodiments, atleast 98% or more. The degree of homology will vary with the length andnumber of BACE polypeptides compared.

It may also be desirable to add amino acids at the amino- orcarboxy-terminus of a BACE polypeptide, e.g., to prepare a fusionprotein. For example, the addition may be a polyhistidine tag of 5-20amino acids, preferably 6 amino acids, in length. Alternatively, ahistidine tag for aiding in purification of a BACE polypeptide may belocated at the carboxy-terminus. Further, a myc tag may be added to thecarboxy-terminus of BACE. The myc tag may be used for detection orimmunopurification of BACE. The myc tag and the polyhistidine tag mayboth be located at the carboxy-terminus or amino-terminus in adoubly-tagged BACE.

Purification and Refolding of β-secretase Polypeptide

After being expressed, BACE polypeptide may be purified from inclusionbodies within the host cells. Purification may be performed by any meansknown in the art, such as sucrose gradient centrifugation. However, BACEis preferably purified by the procedure described in example 2 below.

After purification, the BACE polypeptide may need to be refolded.Refolding may be performed by any means known in the art, such asdialysis. However, BACE is preferably refolded according to theprocedure described in example 2 below.

Preferably, reshuffling compounds are added to improve the efficiency ofrefolding. The standard reshuffling solution includes about 1 mM reducedglutathione, about 0.1 mM oxidized glutathione and about 1mM cysteine.However, the ratios of these compounds may be adjusted to furtherincrease the efficiency of refolding by facilitating disulfidereshuffling. Most preferably, the reshuffling conditions include about0.5 mM reduced glutathione, about 0.5 mM oxidized glutathione and about0 mM cysteine.

The pH of the polypeptide solution also affects the efficiency ofrefolding. The BACE polypeptide appears to refold in the pH range of 4to 8.7. Preferably, the pH is either maintained at 8.7 or reduced to 4.0to facilitate refolding.

Once refolded, the BACE preparation may be subjected to anion exchangechromatography for further purification. It may also be desirable tosubject the BACE preparation to standard size exclusion gel filtration.The protein preparation may be further concentrated using standardtechniques. Finally, the preparation is preferably subjected toultracentrifugation, which produces a monodisperse preparation of BACE.The BACE in the resulting supernatant is useful for crystallizationpurposes.

The terms “monodisperse” and “predominantly uniform molecular species”,in reference to BACE, are used interchangeably to indicate that the meanradius of particles comprising BACE varies by less than about 30%,preferably less than about 15%, as determined by, e.g., conventionaldynamic light scattering methods. A monodisperse BACE in solutionpreferably exists in a monomeric form, however, oligomers (e.g., dimers,trimers, tetramers, etc.) may also exist. Such mixtures of BACE havesubunits of a molecular weight of about 45 kDa.

Processing of β-secretase

The BACE polypeptide is processed to remove the propeptide beforecrystallization. The propeptide constitutes approximately the first 50amino acids of the BACE polypeptide, and preferably constitutes aminoacids 22-45 of the native BACE protein.

Processing may be performed by any means known in the art, such as byusing the endoprotease furin. However, the polypeptide is preferablyprocessed by the procedure in example 3 below, using trans cleavageprocessing. The term “trans-cleavage processing” refers to the abilityof one BACE molecule to enzymatically remove the propeptide of anotherBACE molecule. Briefly, BACE polypeptide is exchanged into about 20 mMHepes at about pH 7.5 and about 150 mM NaCl, and then concentrated toabout 5 mg/ml and incubated at about 4° C. for about two weeks. This isthe preferred processing method for producing crystals of BACE for X-raycrystallography. Alternatively, BACE polypeptide is exchanged into about20 mM Hepes at about pH 7.5 and about 150 mM NaCl, and then concentratedto about 15 mg/ml and incubated at about room temperature for about 72hours.

Both of the above trans processing procedures produce a ragged cut. Thismeans that each trans processing procedure cuts the BACE polypeptide(SEQ ID NO: 18) in two separate locations to produce two separateprocessed polypeptides. Preferably, one of the processed polypeptidescomprises the amino acid sequence set forth in SEQ ID NO: 20.Alternatively, the other processed polypeptide preferably comprises theamino acid sequence set forth in SEQ ID NO: 22.

Enzymatic Activity of Refolded β-secretase

The enzymatic activity of the refolded and processed BACE polypeptidemay be tested in order to assess the functionality of the expressedpolypeptide. The term “enzymatically active” means a polypeptide iscatalytically active and, preferably, can hydrolyze a peptide bond of asuitable substrate. Preferably, the term relates to the ability of BACEto cleave β-amyloid precursor protein or a fragment thereof. Enzymaticactivity may be measured by any means known in the art, such as byquantitating the rates of peptide or protein hydrolysis. However,enzymatic activity is preferably measured by the procedure in example 4below. Unexpectedly, the polypeptides comprising the nucleotidesequences set forth in either SEQ ID NO: 1 or SEQ ID NO: 17 exhibitsenzymatic activity similar to BACE from other expression systems.

The term “active site”, when referring to a BACE polypeptide, describesthe area of the polypeptide responsible for peptide recognition and/orpeptide bond hydrolysis. An active site in an “open configuration” meansthat the active site is accessible to interaction with a suitablesubstrate and/or inhibitor. Preferably, BACE polypeptide is made in asystem which produces BACE with an active site in the openconfiguration.

Crystallization

Embodiments of the invention relate to methods for growing crystals ofBACE. Proteins are crystallized in a crystallization solution. Acrystallization solution preferably contains the protein of interest, aprecipitant, a salt, a buffering agent and, optionally, a reducingagent, oxygen scavenger, protein stabilizing agent or detergent.

For crystallization of BACE, it is desirable to use a solution ofprocessed BACE polypeptide having a concentration ranging from about 1mg/ml to the upper limit of how high the protein can be concentrated insolution. Preferably, the concentration of BACE is about 10 mg/ml toabout 20 mg/ml. More preferably, the BACE concentration is about 14mg/ml to about 17 mg/ml. Most preferably, the BACE concentration isabout 16 mg/ml. Preferably, the solution of processed BACE polypeptidecomprises a mixture of two polypeptides comprising the amino acidsequences set forth in SEQ ID NO: 20 and SEQ ID NO: 22. Alternatively,the solution of processed BACE polypeptide comprises a polypeptidecomprising the amino acid sequence set forth in either SEQ ID NO: 20 orSEQ ID NO: 22.

A “precipitant” is a compound that decreases the solubility of apolypeptide in a concentrated solution. Alternatively, the term“precipitant” can be used to refer to a change in physical or chemicalparameters which decrease polypeptide solubility, including temperature,pH and salt concentration. Precipitants induce crystallization byforming an energetically unfavorable precipitant-depleted layer aroundthe polypeptide molecules. To minimize the relative amount of thisdepletion layer, the polypeptides form associations and, ultimately,crystals. This process is explained in more detail in Weber, Advances inProtein Chemistry 41:1-36 (1991). Various precipitants are known in theart and include, but are not limited to, ammonium sulfate, ethanol,3-ethyl-2,4 pentanediol, and many of the polyglycols, such aspolyethylene glycol.

Crystallization of BACE is preferably achieved in a precipitant solutioncontaining polyethylene glycol 1000-20,000 (PEG; average molecularweight ranging from about 1000 to about 20,000 Da). Most preferably, thepolyethylene glycol is PEG3350 (Hampton Research, Laguna Niguel,Calif.). Preferably, PEG3350 is present in a concentration ranging fromabout 13.75% to about 25% (w/v). More preferably, the concentration ofPEG3350 ranges from about 13.75% to about 15% (w/v). The most preferablePEG3350 concentration is about 15% (w/v). It should be noted thatPEG3350 seems to be the same as PEG4000. The name of the compoundappears to depend upon the manufacturer.

The crystallization solution also contains a salt. Salts act as aco-precipitant because they are used to reduce the solubility of thepolypeptide in solution. Examples of salts include, but are not limitedto, sodium chloride, lithium chloride, sodium citrate, ammonium iodide,ammonium tartrate, Na+/K+ tartrate or any of the tartrate salts. A saltis preferably added to the crystallization solution in a concentrationranging from about 1 mM to about 1000 mM. Preferably, the salt isammonium tartrate or Na+/K+ tartrate, in a concentration of about 0.2 Mto about 0.4 M. Alternatively, a preferred salt is ammonium iodide, in aconcentration ranging from about 0.1 M to about 1 M. More preferably,the concentration of ammonium iodide is 0.6 M or 0.8 M. Most preferably,the concentration of ammonium iodide is 0.6 M.

In addition, buffering agents or buffers are added to thecrystallization solution to adjust the pH of the solution, and hencesurface charge on the polypeptide. The pH of the buffering agent mayrange from about 4 to about 10, e.g., 5, 6, 7, 8 and 9, preferablybetween about pH 7 and about pH 8, e.g., 7.2, 7.4, 7.5, 7.6 and 7.8.Buffers are well known in the art and many are useful in the precipitantsolution (Scopes, Protein Purification: Principles and Practice, Thirded., (1994) Springer-Verlag, New York). Examples of buffers include, butare not limited to, Hepes, Tris, MES and acetate.

Reducing agents may also be added to the crystallization solution.Examples of suitable reducing agents for crystallization include, butare not limited to, dithiothreitol (DTT), dithioerythritol (DET) andβ-mercaptoethanol (BME). If desired, the reducing agent is present inthe solution at a concentration of about 10 mM. Preferably, the BACEcrystallization solution does not include a reducing agent.

In addition, oxygen scavengers may also be added to the crystallizationsolution. Oxygen scavengers are well known in the art and any may beused. Preferably, the BACE crystallization solution does not include anoxygen scavenger.

Protein stabilizers may also be added to the crystallization solution.Over time, proteins in solution have a natural tendency to becomeunfolded. Protein stabilizers prevent denaturation of the protein, andhence, precipitation of the protein in solution. Protein stabilizers arewell known in the art. A preferred protein stabilizer is glycerol. Ifglycerol is chosen as the protein stabilizing agent, it is preferablyprovided at a concentration ranging from about 0.5% to about 20% (w/v).Preferably, the BACE crystallization solution does not include a proteinstabilizer.

Detergents may also be added to the crystallization solution. Proteinsin solution have a natural tendency to react with each other. Detergentsprevent the protein from interacting with itself and with other proteinmolecules in solution. Detergents are well known in the art. Preferably,the BACE crystallization solution does not include a detergent.

Furthermore, other additives may be added to the crystallizationsolution. Examples of these other additives include, but are not limitedto, ethanol and spermidine. A more complete list of additives can befound in the product catalog from Hamptom Research (Laguna Niguel,Calif.).

Crystallization may be accomplished by any of the known techniques inthe art (Gieg6, et al., (1994) Acta Crystallogr. D50: 339-350;McPherson, (1990) Eur. J. Biochem. 189: 1-23). Such techniques include,but are not limited to, microbatch, hanging drop vapor diffusion,seeding and dialysis. Preferably, hanging drop vapor diffusion(McPherson, (1976) J. Biol. Chem. 251: 6300-6303) or microbatch methods(Chayen (1997) Structure 5: 1269-1274) are used. Most preferably,crystallization is performed using hanging drop vapor diffusion. In eachof these methods, it is important to promote continued crystal growthafter nucleation by maintaining a supersaturated solution.

In hanging drop vapor diffusion, a protein of interest (in water) issolubilized in a drop of crystallization solution and placed on asubstrate, such as a microscope slide. The substrate is then turned overso the drop hangs from the substrate. The surface tension in the dropkeeps the drop from falling due to the forces of gravity. The substrateand drop are then placed over a pool of crystallization solution. Thesystem is then sealed. Over time, the two solutions equilibrate bydiffusion, causing the protein to crystallize. In the microbatch method,polypeptide is mixed with precipitants to achieve supersaturation, andthe vessel is then sealed and set aside until crystals appear. In thedialysis method, polypeptide is retained in a sealed dialysis membranewhich is placed into a solution containing precipitant. Equilibrationacross the membrane increases the precipitant concentration, therebycausing the polypeptide to reach supersaturation levels.

Crystals routinely grow in a wide range of temperatures. It is, however,preferred to grow crystals by the hanging drop method at temperaturesbetween about 2° C. and about 26° C., more preferably between about 2°C. to about 8° C., and most preferably at about 4° C.

Crystals of BACE may be grown in either the uncrystallized or apo form,without a bound ligand, or, alternatively, complexed to a ligand,preferably an inhibitor. Each crystal form of BACE (uncomplexed form orcomplexed form) is useful because both crystal forms can be used togather knowledge about the structure of BACE and potential ligands ofBACE. BACE can be complexed with any ligand, such as OM-99-2 (SEQ ID NO:15), to form a crystal.

In a preferred embodiment, uncomplexed, refolded processed BACE wascrystallized. The crystallization procedures are described in moredetail in example 6 below. Briefly, crystallization was carried out atabout 4° C. using the hanging drop method. The optimal crystallizationconditions included about 13.75% to about 15% PEG3350 and about 0.6 Mammonium iodide at about 4° C.

In another preferred embodiment, refolded processed BACE in the presenceof OM-99-2 (SEQ ID NO: 15) was crystallized. The crystallizationprocedures are described in more detail in example 5 below. Briefly,crystallization was carried out at about 4° C. using the hanging dropmethod. The optimal crystallization conditions included about 20%PEG3350 and about 0.2 M ammonium tartrate at about 4° C.

Embodiments of the present invention also include crystals comprisingBACE polypeptide as disclosed by Vassar et al., (1999) Science, 286:735-741-Genbank Accession No. AF190725; Murphy et al., (2001)Neuroreport, 12(3):631-634; Capell et al., (2000) J. Biol. Chem.,275(40):30849-30854 and Haniu et al., (2000) J. Biol. Chem.,275(28):21099-21106.

Crystallographic Analysis

The crystals of the present invention have a variety of uses. Forexample, high quality crystals are suitable for X-ray or neutrondiffraction analysis, which can be used to determine thethree-dimensional structure of BACE, and, in particular, to assist inthe identification of the protein's active and effector sites. Knowledgeof these sites and solvent accessible residues allow for structure-baseddesign and construction of ligands, agonists and antagonists for BACE.

In addition, crystallization itself can be used as a purificationmethod. In some instances, a polypeptide crystallizes from aheterogeneous mixture. Isolation of such crystals, by filtration and/orcentrifugation, followed by redissolution of the polypeptide provides apurified solution suitable for use in growing high-quality crystals,which are preferred for diffraction analysis.

Once a crystal of a polypeptide or protein is grown, the crystal isfrozen so that X-ray diffraction data of the crystal can be collected. Acrystal of a protein may be frozen by any means in the art. In addition,the freezing process may occur in one step or in several steps.Preferably, the BACE crystal is frozen in two steps. In the first step,the crystal is frozen in a solution including about 20% PEG3350, about0.48 M ammonium iodide, and about 15% glycerol. In the second step, thecrystal is frozen in a solution including about 20% PEG3350, about 0.48M ammonium iodide, and about 20% glycerol.

One method for determining the three-dimensional structure of a proteinfrom X-ray diffraction data of a protein crystal includes the use ofsynchrotron radiation, under standard cryogenic conditions. However,alternative methods may also be used. For example, crystals may becharacterized using X-rays produced by a conventional source, such as asealed tube or a rotating anode. Methods of characterization include,but are not limited to, precession photography, oscillation photographyand diffractometer data collection.

Preferably, the crystals or the soluble polypeptides which are used toform the crystals exhibit BACE catalytic activity (see above). Mostpreferably, the BACE crystals include a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 22, which isderived from the nucleotide sequence set forth in SEQ ID NO: 1 andexpressed in E. coli host cells.

An embodiment of the invention provides crystals of BACE polypeptide inthe uncomplexed form. Preferably, the BACE polypeptide is derived fromhumans. Preferably, the BACE polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 22. More preferably, the BACEpolypeptide is derived from the nucleotide sequence set forth in SEQ IDNO: 1. Preferably, the BACE crystals effectively diffract X-rays for thedetermination of the atomic coordinates of BACE to a resolution greaterthan about 5.0 Å.

Still another embodiment of the invention provides a method for using acrystal of the present invention to obtain detailed three-dimensionalstructural data and coordinates for uncomplexed BACE, using X-raycrystallography. Preferably, the crystals of uncomplexed BACE arecharacterized by the structure coordinates set forth in Table 1. Morepreferably, the crystals of uncomplexed BACE have a space group of C2with unit cell dimensions of a=236.0 Å, b=103.6 Å, and c=65.0 Å.However, the unit cell dimension values for a, b and c may vary by ±2%.Therefore, the crystals of uncomplexed BACE may have a space group of C2with unit cell dimensions wherein the value for a may range from about231.3 Å to about 240.7 Å, the value for b may range from about 101.5 Åto about 105.7 Å, or the value for c may range from about 63.7 Å toabout 66.3 Å.

Another embodiment of the invention provides crystals of aprotein-ligand complex comprising BACE and a ligand. Preferably, theBACE polypeptide is derived from humans. Preferably, the BACEpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:22. More preferably, the BACE polypeptide is derived from the nucleotidesequence set forth in SEQ ID NO: 1. Preferably, the BACE crystalseffectively diffract X-rays for the determination of the atomiccoordinates of BACE to a resolution greater than about 5.0 Å.

Yet still another embodiment of the present invention provides a methodfor using a crystal of the present invention to obtain detailedthree-dimensional structural data and coordinates for a protein-ligandcomplex comprising BACE and a ligand, using X-ray crystallography.Preferably, the crystals of complexed BACE are characterized by thestructure coordinates set forth in Table 2. More preferably, thecrystals of complexed BACE have a space group of P2₁2₁2₁ with unit celldimensions of a=86.4 Å, b=89.1 Å, and c=131.3 Å. However, the unit celldimension values for a, b and c may vary by ±2%. Therefore, the crystalsof complexed BACE may have a space group of P2₁2₁2₁ with unit celldimensions wherein the value for a may range from about 84.7 Å to about88.1 Å, the value for b may range from about 87.3 Å to about 90.9 Å, orthe value for c may range from about 128.7 Å to about 133.9 Å.

The crystallizable compositions of the present invention are preferablyamenable to X-ray crystallography for providing the three-dimensionalstructure of a BACE polypeptide. Embodiments of the present inventioninclude crystals which effectively diffract X-rays for a determinationof the atomic coordinates of BACE to a resolution of greater than about5.0 Ångströms, e.g., about 4.5 Å, about 4 Å, about 3 Å, about 2.5 Å,about 2 Å, about 1 Å, about 0.5 Å, about 0.1 Å, preferably greater thanabout 4.0 Ångströms, e.g., about 3 Å, about 2.5 Å, about 2 Å, about 1 Å,about 0.5 Å, about 0.1 Å, more preferably greater than about 2.8Ångströms, e.g., about 2.5 Å, about 2.2 Å, about 2 Å, about 1 Å, about0.5 Å, about 0.1 Å, and most preferably greater than about 2.0Ångströms, e.g., about 1.7 Å, about 1.5 Å, about 1 Å, about 0.5 Å, about0.1 Å.

As described above, embodiments of the present invention include BACEcrystals whose three-dimensional structures are described by thestructure coordinates set forth in either Table 1 or Table 2. Likewise,embodiments of the present invention also include crystals that possessstructure coordinates which are structurally similar to those set forthin either Table 1 or Table 2. Structural similarity between crystals isdiscussed in detail below. The term “structure coordinates” refers toCartesian coordinates derived from mathematical equations related to thepatterns obtained upon diffraction of a beam of X-rays by the atoms(scattering centers) of a molecule. The diffraction data are used tocalculate electron density maps and to establish the positions of theindividual atoms of the molecule.

Those of skill in the art will understand that a set of structurecoordinates for a polypeptide or a protein-ligand complex or a portionthereof is a relative set of points that define a shape in threedimensions. Thus, it is possible that an entirely different set ofcoordinates could define a similar or identical shape. Moreover, slightvariations in the individual coordinates will have little effect onoverall shape.

Embodiments of the present invention include crystals exhibitingstructure coordinates which are structurally similar to those set forthin either Table 1 or Table 2, but for crystallographic permutations ofthe structure coordinates, fractionalization of the structurecoordinates, additions, subtractions, rotations or translations to setsof the structure coordinates, or any combinations of the above.

Alternatively, modifications in the crystal structure due to mutations,additions, substitutions, and/or deletions of amino acids, or otherchanges in any of the components that make up the crystal may alsoaccount for variations in the structure coordinates. If such variationsare within an acceptable standard error, as compared to the structurecoordinates set forth in either Table 1 or Table 2, the resultingthree-dimensional shape is considered to be the same and, accordingly,the modified crystal is considered to be within the scope of the presentinvention.

Various computational analyses may be necessary to determine whether acrystal is sufficiently similar to the crystals whose structurecoordinates are set forth in either Table 1 or Table 2 as to beconsidered the same. Such analyses may be carried out in currentsoftware applications, such as the Molecular Similarity application ofQUANTA (Accelyris, San Diego, Calif.) version 4.1, and as described inthe accompanying User's Guide.

The Molecular Similarity application permits comparisons betweendifferent structures, different conformations of the same structure, anddifferent parts of the same structure. In general, the procedure used inMolecular Similarity to compare structures is divided into foursteps: 1) input the structures to be compared; 2) define the atomequivalences in these structures; 3) perform a fitting operation; and 4)analyze the results.

Each structure is identified by a name. One structure is identified asthe target, i.e., the fixed structure; all remaining structures areworking structures, i.e., moving structures. Because atom equivalencywithin QUANTA is defined by user input, for the purpose of thisapplication, we will define equivalent atoms as protein backbone atoms(N, Cα, C and O) for all conserved residues between the two structuresbeing compared.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses a least squares fitting algorithm that computesthe optimum translation and rotation to be applied to the movingstructure, such that the root mean square difference of the fit over thespecified pairs of equivalent atoms is an absolute minimum. This number,given in Ångströms, is reported by QUANTA.

The term “root mean square deviation” (RMSD) is a common term in the artwhich, in general, means the square root of the arithmetic mean of thesquares of the deviations from the mean distance of corresponding atoms.It is a way to express the deviation or variation from a trend orobject. The term “least squares” refers to a method based on theprinciple that the best estimate of a value is that in which the sum ofthe squares of the deviations of observed values is a minimum.

For the purpose of this application, any set of structure coordinates ofa molecule that has a RMSD of conserved residue backbone atoms (N, Cα,C, O) of less than about 1.5 Å when superimposed—using backbone atoms—onthe relevant structure coordinates set forth in either Table 1 or Table2 are considered identical and are within the scope of the presentinvention. Preferably, the root mean square deviation is less than about1.0 Å. More preferably, the root mean square deviation is less thanabout 0.5 Å. Most preferably, the root mean square deviation is lessthan about 0.1 Å.

In a preferred embodiment of the invention, crystallographic analysis ofuncomplexed, refolded, processed BACE comprising a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 22, which wasderived from the nucleotide sequence set forth in SEQ ID NO: 1 andexpressed in E. coli cells, was performed. The crystallographic analysisprocedures are described in more detail in example 8 below. Briefly, theuncomplexed BACE crystals of example 6 were transferred to a solutioncontaining about 20% PEG3350, about 0.6 M ammonium iodide and about 15%glycerol, and then frozen in liquid propane. Diffraction data werecollected. Data reduction showed diffraction to about 2.2 Å resolution.The crystals had the space group of C2 with unit cell dimensions ofa=236.0 Å, b=103.6 Å, and c=65.0 Å. The crystals may be characterized bythe structure coordinates set forth in Table 1.

In another preferred embodiment of the invention, crystallographicanalysis of refolded, processed BACE comprising a polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 22, which was derivedfrom the nucleotide sequence set forth in SEQ ID NO: 1 and expressed inE. coli cells, complexed to OM-99-2 (SEQ ID NO: 15) was performed. Thecrystallographic analysis procedures are described in more detail inexample 7 below. Briefly, the BACE-inhibitor complex crystals of example5 were transferred to a solution containing about 22% PEG3350, about 0.2M ammonium tartrate and about 15% PEG400, and then frozen in liquidpropane. Diffraction data were collected. Data reduction showeddiffraction to about 1.7 Å resolution. The crystals had the space groupof P2₁2₁2₁, with unit cell dimensions of a=86.4 Å, b=89.1 Å, and c=131.3Å. The crystals may be characterized by the structure coordinates setforth in Table 2.

Uses of Crystals and/or Structure Coordinates

An embodiment of the invention provides a computer comprising thestructure coordinates set forth in Table 1. Another embodiment of theinvention provides a computer comprising the structure coordinates setforth in Table 2.

In accordance with an embodiment of the invention, the structurecoordinates of BACE polypeptide and portions thereof may also be storedin a machine-readable data storage medium. Such data may be used for avariety of purposes, such as drug discovery and X-ray crystallographicanalysis of a protein crystal, e.g., for producing a three-dimensionalrepresentation of BACE. Accordingly, embodiments of the inventionprovide machine-readable magnetic data storage media comprising a datastorage material encoded with the structure coordinates set forth ineither Table 1 or Table 2. The machine-readable magnetic data storagemedium may also include any set of structure coordinates of a moleculethat has a root mean square deviation of conserved residue backboneatoms (N, Cα, C, O) of less than about 1.5 Å, preferably, less thanabout 1.0 Å, more preferably less than about 0.5 Å, and most preferablyless than about 0.1 Å when superimposed—using backbone atoms—on therelevant structure coordinates set forth in either Table 1 or Table 2.

A computer system, useful in reading the machine readable data storagemedium, including a computer comprising a central processing unit (CPU)and a memory storage device, is also within the scope of the presentinvention. In general, the computer system may be any computer with anoperating system, such as MS-DOS, PC-DOS, Windows, OS/2, Unix, Unixvariant or MacOS. Particularly preferred computer systems are theSilicon Graphics Octane workstation or Compaq AlphaServer DS20. Otherhardware systems and software packages are known to those skilled in theart.

Input hardware coupled to the computer system by input line, may beimplemented in a variety of ways. Machine-readable data may be inputtedvia the use of a modem or modems connected by a telephone line or adedicated data line. Alternatively, or additionally, the input hardwaremay comprise CD-ROM drives or disk drives. A keyboard may also be usedas an input device.

Output hardware, coupled to the computer system by output lines, maysimilarly be implemented by conventional devices. By way of example,output hardware may include a display terminal, e.g., a cathode ray tube(CRT), for displaying a graphical representation of thethree-dimensional structure of BACE or a portion thereof using a programsuch as INSIGHT (Accelyris, San Diego, Calif.) or QUANTA as describedherein. Output hardware might also include a printer, so that hard copyoutput may be produced, or a disk drive, to store system output forlater use. In preferred embodiments, the computer possesses a displaywhich is displaying a three-dimensional representation of BACE or afragment or homologue thereof.

In operation, the central processing unit (CPU) coordinates the use ofthe various input and output devices, coordinates data access from massstorage and access to and from working memory, and determines thesequence of data processing steps. A number of programs may be used toprocess the machine-readable data. Such programs are discussed inreference to the computational methods of drug discovery, as describedherein. Specific references to components of the computer system areincluded, as appropriate, throughout the following description of thedata storage medium.

A magnetic data storage medium can be encoded with machine-readable databy a computer system, as described above. The storage medium may be, forexample, a conventional floppy diskette or hard disk, having a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, on one or both sides, containing magnetic domains whosepolarity or orientation can be altered magnetically. The magneticdomains of the coating of medium may be polarized or oriented so as toencode, in a manner which may be conventional, machine readable data,such as that described herein, for execution by a system as describedherein. The storage medium may also have an opening for receiving thespindle of a disk drive or other data storage device. Alternatively, anoptically-readable data storage medium can be encoded with suchmachine-readable data, or a set of instructions. The medium can be aconventional compact disk read only memory (CD-ROM), or a rewritablemedium, such as a magneto-optical disk, which is optically readable andmagneto-optically writable, or a CDRW.

In general, in the case of a CD-ROM, as is well known, the disk coatingis reflective and is impressed with a plurality of pits to encode themachine-readable data. The arrangement of the pits is read by reflectinglaser light off the surface of the coating. A protective coating, whichpreferably is substantially transparent, is provided on top of thecoating.

In general, in the case of a magneto-optical disk, as is well known, thedisk coating does not have pits, but has a plurality of magnetic domainswhose polarity or orientation can be changed magnetically when heatedabove a certain temperature, as by a laser. The orientation of thedomains can be read by measuring the polarization of laser lightreflected from the coating. The arrangement of the domains encodes thedata, as described above.

An embodiment of the present invention provides the use ofstructure-based drug design techniques to design, select, and synthesizechemical entities, including inhibitory compounds, that are capable ofbinding to BACE. Also, embodiments of the present invention provide denovo and iterative drug design methods that can be used to develop drugsfrom the structures of the BACE crystals of the present invention.

One particularly useful structure-based drug design technique enabled bythe present invention is rational drug design. Rational drug design is amethod for optimizing associations between a polypeptide and a ligand bydetermining and evaluating the three-dimensional structures ofsuccessive sets of protein/ligand complexes. The ligand can be any sortof compound, including, but not limited to a chemical, polypeptide, ormodified polypeptide.

Those skilled in the art will appreciate that association of naturalligands or substrates with the binding pockets of their correspondingreceptors or enzymes is the basis of many biological mechanisms ofaction. The term “binding pocket”, as used herein, may refer to anyregion of a molecule or molecular complex, that, as a result of itsshape, favorably associates with another chemical entity or compound.Similarly, drugs may exert their biological effects through associationwith the binding pockets of receptors and enzymes. Such association mayoccur with all or any part of the binding pockets.

An understanding of such associations will help lead to the design ofdrugs having more favorable associations with the target enzyme, andthus, improved biological effects. For example, associations between apolypeptide and ligand are optimized by filling the space in the bindingpocket between the polypeptide and the ligand, yet not allowing theligand to overlap the polypeptide. Therefore, information about whereand how to alter a ligand to achieve increased binding, increasedpotentcy, etc., is obtained. Preferably, this analysis is performed inconjunction with computer modeling, i.e., the use of computers tovisualize and aid in understanding the associations between apolypeptide and a ligand. Therefore, this information is valuable indesigning potential enzyme ligands, such as inhibitors of BACE.

In iterative structure-based drug design, crystals of a series ofprotein/ligand complexes are obtained and then the three-dimensionalstructure of each complex is solved. Such an approach provides insightinto the association between the proteins and ligands of each complex.This may be accomplished by selecting ligands that bind to the protein,obtaining crystals of a new complex, solving the three-dimensionalstructure of the complex, and comparing the associations between the newcomplex and previously solved complex. By observing how changes in theligand affected the protein/ligand associations, these associations maybe optimized. Preferably, this is performed in conjunction with computermodeling.

In some cases, iterative structure-based drug design is carried out byforming successive protein/ligand complexes and then crystallizing eachnew complex. This method can be time consuming because it takesapproximately 7-21 days to grow a crystal. Alternatively, a pre-formedprotein crystal may be soaked in the presence of a ligand, therebyforming a protein/ligand complex and obviating the need to crystallizeeach individual protein/ligand complex. This process usually only takesabout 1 day to perform because the crystal is already formed. As usedherein, the term “soaked” refers to a process in which the crystal istransferred to a solution containing the ligand of interest. Anothermethod involves using a co-crystal, a crystal of a protein bound to afirst ligand. The co-crystal is then soaked in the presence of apotential ligand. The potential ligand then displaces the first ligandfrom the crystal. Similarly, this process usually takes about 1 day toperform. Advantageously, BACE crystals provided by this invention may besoaked in the presence of a ligand, such as BACE inhibitors, substratesor other ligands to provide novel BACE/ligand crystal complexes.

For example, a preferred embodiment of the invention provides a methodfor identifying a ligand that binds to β-secretase. In this embodiment,about 16 mg/ml of β-secretase comprising the amino acid sequence setforth in SEQ ID NO: 22 is added to about 1.5 to about 5.0 molar ratio ofa ligand to form a mixture. Preferably, the β-secretase is derived fromhuman. More preferably, the β-secretase is expressed in E. coli cellsand derived from the nucleotide sequence set forth in SEQ ID NO: 1. Themixture is then crystallized to form a crystal; and X-ray diffractionanalysis is performed on the crystal.

Another preferred embodiment of the invention provides a method foridentifying a ligand that binds to β-secretase comprising soaking a BACEcrystal in a solution comprising a ligand and performing X-raydiffraction on the crystal. Preferably, the crystal is made by addingabout 16 mg/ml of a polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 22 to a crystallization solution, the solutioncomprising about 13.75% to about 15% PEG3350 and about 0.6 M ammoniumiodide, and crystallizing the solution at about 4° C. using a hangingdrop method. Preferably, the β-secretase is derived from human. Morepreferably, the β-secretase is expressed in E. coli cells and derivedfrom the nucleotide sequence set forth in SEQ ID NO: 1.

Yet another preferred embodiment of the invention provides anothermethod for identifying a ligand that binds to β-secretase. Thisembodiment involves preparing a mixture of β-secretase with a potentialligand comprising adding a first ligand to about 16 mg/ml of β-secretasecomprising the amino acid sequence set forth in SEQ ID NO:

22; crystallizing the mixture to form a crystal; soaking the crystal ina solution comprising a potential ligand, wherein the potential liganddisplaces the first ligand from the crystal; and performing X-raydiffraction on the crystal. Preferably, the β-secretase is derived fromhuman. More preferably, the β-secretase is expressed in E. coli cellsand derived from the nucleotide sequence set forth in SEQ ID NO: 1.

Another embodiment of the invention provides a method for identifying aligand that binds to β-secretase by obtaining a set of atomiccoordinates defining the three-dimensional structure of a crystal of anuncomplexed, processed β-secretase polypeptide expressed in E. coli thateffectively diffracts X-rays for determination of the atomic coordinatesof the β-secretase to a resolution of greater than about 5.0 Å;selecting a ligand by performing rational drug design with the set ofatomic coordinates obtained above; contacting the ligand to theβ-secretase; and detecting binding of the ligand to the β-secretase.Preferably, the selection is performed in conjunction with computermodeling.

The extent of binding may be determined by a standard binding assay. Forexample, a substrate of BACE, such as APP, may be attached to a solidsupport. Methods for attaching polypeptides to solid supports are knownin the art. The substrate may then be labeled. The solid support may bewashed to remove unreacted species. A solution containing BACE and/orpotential inhibitor may then be contacted to the support. The solidsupport may then be washed again to remove any fragments of thesubstrate that were cleaved by BACE. The amount of labeled substrateremaining on the solid support may then be determined.

Alternative embodiments of the invention provide methods for identifyinginhibitors, or antagonists, of β-secretase. An embodiment of theinvention provides a method for identifying a β-secretase antagonistcomprising the steps of: (a) selecting a potential antagonist byperforming rational drug design using the three-dimensional structure ofa crystal of a β-secretase polypeptide; (b) contacting the potentialantagonist with β-secretase; and (c) detecting binding of the potentialantagonist to the β-secretase, wherein an antagonist is identified onthe basis of its ability to inhibit the catalytic activity of theβ-secretase. Preferably, the β-secretase is expressed in E, coli cells.Preferably, the β-secretase comprises a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 22. It is also preferable that thecrystal effectively diffracts X-rays for a determination of atomiccoordinates of the polypeptide to a resolution of greater than about 5.0Å.

Another embodiment of the invention provides a method for identifying aninhibitor of β-secretase comprising: (a) obtaining a set of atomiccoordinates from a crystal defining the three-dimensional structure of aβ-secretase polypeptide; (b) selecting a potential inhibitor byperforming rational drug design with the set of atomic coordinatesobtained above; (c) contacting the potential inhibitor with aβ-secretase protein; and (d) measuring the activity of the protein,wherein the potential inhibitor is identified when there is a decreasein activity of the β-secretase in the presence of the inhibitor ascompared to the activity of β-secretase in the absence of the potentialinhibitor. Preferably, the β-secretase polypeptide is uncomplexed andexpressed in E. coli cells. In addition, it is preferable that theβ-secretase comprises a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 22. Furthermore, it is preferable to perform theselecting step in conjunction with computer modeling. Most preferably,the method provides a crystal having a space group of C2 with unit celldimensions of a=236.0 Å, b=103.6 Å and c=65.0 Å. Alternatively, themethod may provide a crystal having a space group of C2 with unit celldimensions wherein a ranges from about 231.3 Å to about 240.7 Å, branges from about 101.5 Å to about 105.7 Å, and c ranges from about 63.7Å to about 66.3 Å.

A further embodiment of the invention provides a method for identifyinga potential inhibitor of β-secretase comprising the steps of: (a)viewing a three-dimensional structure of the β-secretase; (b) employingthe three-dimensional structure to design or select the potentialinhibitor; (c) synthesizing the potential inhibitor; and (d) contactingthe potential inhibitor with the β-secretase in the presence of asubstrate to determine the ability of the potential inhibitor to inhibitthe β-secretase. Preferably, the β-secretase is defined by the atomiccoordinates set forth in Table 1.

Another aspect of the invention is the use of the structure coordinatesand atomic details of BACE or mutants or homologues or co-complexesthereof to design, evaluate computationally, synthesize and useinhibitors (antagonists) of BACE that prevent or treat the undesirablephysical and pharmacological properties of Alzheimer's Disease. Theseinhibitors (antagonists) may be used in the treatment of Alzheimer'sDisease.

In an embodiment of the invention, the structure coordinates set forthin either Table 1 or Table 2 may be used to aid in obtaining structuralinformation about another crystallized molecule or molecular complex.This may be achieved by any of a number of well-known techniques,including molecular replacement.

In another embodiment of the invention, the structure coordinates setforth in either Table 1 or Table 2 may also be used for determining atleast a portion of the three-dimensional structure of molecules ormolecular complexes which contain at least some structurally similarfeatures to BACE. In particular, structural information about anothercrystallized molecule or molecular complex may be obtained by well-knowntechniques, including molecular replacement.

Therefore, embodiments of the invention provide methods for utilizingmolecular replacement to obtain structural information about acrystallized molecule or molecular complex, whose structure is unknown,comprising the steps of generating an X-ray diffraction pattern from thecrystallized molecule or molecular complex and applying crystallographicphases derived from at least a portion of the structure coordinates setforth in either Table 1 or Table 2 to the X-ray diffraction pattern togenerate a three-dimensional electron density map of the molecule ormolecular complex whose structure is unknown.

Once the structure coordinates of a protein crystal have beendetermined, they are useful in solving the structures of other crystals.In addition, the structure of BACE homologues may be determined from thestructure coordinates of the present invention. For example,polypeptides may be crystallized and their structure elucidated by, forexample, difference Fourier techniques and molecular replacement.

By using molecular replacement, all or part of the structure coordinatesof a BACE polypeptide provided by this invention, and set forth ineither Table 1 or Table 2, can be used to determine the previouslyunknown structure of a crystallized molecule or molecular complex morequickly and efficiently than attempting to determine such information abinitio.

Molecular replacement provides an accurate estimation of the phases foran unknown structure. Phases are a factor in equations used to solvecrystal structures that cannot be measured experimentally. Obtainingaccurate values for the phases, by methods other than molecularreplacement, is a time-consuming process. However, when the crystalstructure of a protein containing a homologous portion has been solved,the phases from the known structure may provide a satisfactory estimateof the phases for the unknown structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex, whose structure coordinates are unknown, byorienting and positioning the relevant portion of the BACE crystalaccording to either Table 1 or Table 2 within the unit cell of thecrystal of the unknown molecule or molecular complex so as to bestaccount for the observed X-ray diffraction pattern amplitudes togenerate an electron density map of the structure whose coordinates areunknown. This, in turn, can be subjected to any well-known modelbuilding and structure refinement techniques to provide a final,accurate structure of the unknown crystallized molecule or molecularcomplex (Lattman, “Use of the Rotation and Translation Functions”, inMeth. Enzymol., 115: 55-77 (1985); Rossman, ed., “The MolecularReplacement Method”, Int. Sci. Rev. Ser., No. 13, Gordon & Breach, NewYork (1972)).

Phase information from the structure coordinates of the presentinvention may be used to elucidate the structure of other crystals. Forexample, the structure of BACE in complex with other atoms or moleculesmay be elucidated. Such complexes include, for example, those containingatoms soaked into or co-crystallized within the crystal lattice. Otherstructures which can be elucidated using the phase information of thepresent invention include, for example, other proteases or homologues,or mutants thereof, having sufficient three-dimensional structuresimilarity to BACE complex as to be solved using molecular replacement.Examples of such proteins include, but are not limited to, cathepsin D,renin and pepsin. Also, these protein molecules in a complex with asmall molecule substrate(s), inhibitor(s), transition state analog(s),product(s) or analog(s) of any of these may also be solved using thephase information of the present invention. Other complexes whosestructure can be elucidated from the phase information of the presentinvention include BACE complexed with an inhibitor. Complexes containinga combination of the above molecules may also be solved using the phaseinformation of the present invention.

The structure of any portion of any crystallized molecule or molecularcomplex that is sufficiently homologous to any portion of the BACEprotein can be solved by this method. The difference Fourier methodsimply calculates an electron density map using phases calculated fromthe structure coordinates and observed diffraction amplitudes from acrystal of an unknown structure. This method is often used to solvestructures of protein/ligand complexes where the ligand is small anddoes not affect the crystal form significantly.

In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about a molecule wherein themolecule comprises a BACE polypeptide complex. The structure coordinatesof BACE provided by this invention are particularly useful in solvingthe structure of other crystal forms of BACE polypeptide complexes. Thisapproach enables the determination of the optimal sites for interactionbetween chemical entities, including interaction of candidate inhibitorswith BACE.

BACE crystals may be studied using well-known X-ray diffractiontechniques and may be refined versus X-ray data to 3 Å resolution orbetter to an R_(free) value of about 0.40 or less using computersoftware such as X-PLOR (Yale University, 1992, distributed by MolecularSimulations, Inc.; see e.g., Blundell & Johnson, supra; Meth, Enzymol.,vol. 114 & 115, H.W. Wyckoff et al., eds., Academic Press (1985)). Thisinformation may be used to optimize known BACE inhibitors and to designnew BACE inhibitors.

EXAMPLES

The present invention may be better understood by reference to thefollowing non-limiting examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate embodiments of the invention, and should in no way beconstrued as limiting the broad scope of the invention.

Example 1 Cloning of β-secretase

Rather than cloning an entire β-secretase gene from an organism, asynthetic optimized nucleic acid sequence of human brain β-secretase wasgenerated. The human brain β-secretase gene was optimized by polymerasechain reaction (PCR) such that the first approximately one-third of thegene was modified.

Human brain β-secretase cDNA contains approximately 70% GC content atthe N-terminus, approximately the first 1-420 bp (base pairs). Thisattribute may be responsible for DNA rearrangement observed duringrecombinant DNA manipulation and overall low expression levels of BACE.Therefore, the sequence analysis programs Oligo 6 (Molecular BiologyInsights, Inc., Cascade, Colo.) and GCG 6 (Genetics Computer Group,version 6, Madison, Wis.) were used to redesign the DNA sequence todecrease the GC content to about 50%, to optimize the codon usage for E.coli expression, and to keep the resulting protein sequence unchangedfrom the native sequence. Decreasing the GC content of the nucleotidesequence reduces the potential for secondary structure formation ofmRNA, which results in decreased levels of protein expression. The codonusage was optimized by using codons that are preferred in E. coli.Preferred codons are determined by sequencing genomic DNA of the hostorganism and applying statistical analysis to determine which codons arepreferred in nature.

A total of 11 oligonucleotides were synthesized and purified bypolyacrylamide gel electrophoresis (PAGE). The oligonucleotides arelisted in the 5′ to 3′ direction. 1) Bwy1F (SEQ ID NO:3)ATGGCTCAAGCTTTGCCATGGTTATTGTTGTGGATGGGTGCTGGTGTTTTACCTGCACATGGTACTCAGCACGGTATCCG 2) Bwy2F (SEQ ID NO:4)TTTACCTTTACGTTCTGGTTTAGGTGGTGCACCATTAGGTTTACGTTTACCTCGTGAGACTGACGAAGAGCCAGA 3) Bwy3F (SEQ ID NO:5)CAGGTCGTCGTGGTTCTTTTGTTGAGATGGTTGACAACTTACGTGGTAAGTCTGGTCAGGGTTACTACGTTGAGATGACT 4) Bwy4F (SEQ ID NO:6)GTTGGTTCTCCACCACAGACTTTAAACATCTTAGTTGATACTGGTTCTTCTAACTTTGCAGTTGGTGCAGCACCACACCC 5) Bwy5F (SEQ ID NO:7)ATTCTTACATCGTTACTACCAGCGTCAGTTATCTTCTACTTACCGTGACTTACGTAAGGGTGTTTATGTTCCAT 6) Bwy6R (SEQ ID NO:8)ACCTAATGGTGCACCACCTAAACCAGAACGTAAAGGTAAACGGATACCGTGCTGAGTACCATGTGCAGGTAAAACACCAGC 7) Bwy7R (SEQ ID NO:9)AAGTTGTCAACCATCTCAACAAAAGAACCACGACGACCTGGCTCCTCTGGCTCTTCGTCAGTCTCACGAGGTAAACGTAA 8) Bwy8R (SEQ ID NO:10)TATCAACTAAGATGTTTAAAGTCTGTGGTGGAGAACCAACAGTCATCTCAACGTAGTAACCCTGACCAGACTTACCACGT 9) Bwy9R (SEQ ID NO:11)AGTAGAAGATAACTGACGCTGGTAGTAACGATGTAAGAATGGGTGTGGTGCTGCACCAACTGCAAAGTTAGAAGAACCAG 10) Bwy10R (SEQ ID NO:12)CTCACCTTCCCACTTACCCTGAGTGTATGGAACATAAACACCCTTACGTA AGTCACGGA 11) Bs10R(SEQ ID NO:13) ACGGATCCTTAGTGGTGGTGGTGGTGGTGGCTCCCTGACTCATCTGTCTGTGGAATGTTGTA

A three-stage PCR strategy was adopted to construct the solublesynthetic β-secretase gene (SEQ ID NO: 17).

In stage 1, two separate half PCR reactions were assembled in two 0.5 mlPCR tubes. Reaction mix 1 contained the primers Bwy1F (SEQ ID NO: 3),Bwy2F (SEQ ID NO: 4), Bwy3F (SEQ ID NO: 5), Bwy6R (SEQ ID NO: 8), Bwy7R(SEQ ID NO: 9), and Bwy8R (SEQ ID NO: 10) at a final concentration of2.5 μM. Reaction mix 2 contained the primers Bwy4F (SEQ ID NO: 6), Bwy5F(SEQ ID NO: 7), Bwy9R (SEQ ID NO: 11), and Bwy10R (SEQ ID NO: 12) at thefinal concentration of 2.5 μM.

Both reaction mixes were heated at 95° C. for 3 minutes, and then cooledto 4° C. at a ramp rate of −5° C./min. Then, using pfu DNA polymerase(Stratagene, La Jolla, Calif.), 25 thermal cycles of PCR were performedat 94° C. for one minute, 55° C. for 1 minute, and 72° C. for oneminute. The reaction generated two half fragments, PM1 and PM2. The twoPCR products were separated using agarose gel electrophoresis andsubsequently purified using Gel Extraction Kit (Qiagen, Valencia,Calif.) according to the manufacturer's instructions.

In stage 2, the synthetic fragment of 1-420 bp of β-secretase (SEQ IDNO: 1) was amplified using the following PCR reaction mix: 50 ng of PM1,50 ng of PM2, 0.2 μM Bwy1F (SEQ ID NO: 3), 0.2 μM Bwy10R (SEQ ID NO:13), 0.5 mM dNTP, 5 μl of 10× reaction buffer, 1 U of pfu DNA polymerase(Stratagene, La Jolla, Calif.), and enough dH₂O to adjust the finalreaction volume to 50 μl. As in stage 1, the mix was heated at 95° C.for 3 minutes, and then cooled to 4° C. at a ramp rate of −5° C./min.Then, using pfu DNA polymerase (Stratagene, La Jolla, Calif.), 25thermal cycles of PCR were performed at 94° C. for one minute, 55° C.for 1 minute, and 72° C. for one minute. The amplified product of thePCR reaction, syn420 (SEQ ID NO: 1), was purified from the agarose gelusing Gel Extraction Kit (Qiagen, Valencia, Calif.).

In stage 3, the synthetic soluble β-secretase, nucleotides 1-1362 bp(SEQ ID NO: 17), was amplified. The PCR reaction mix contained: 50 ng ofsyn420 (SEQ ID NO: 1), 50 ng of β-secretase cDNA (1-1362 bp), 0.2 μMBwy1F (SEQ ID NO: 3), 0.2 μM Bs10R (SEQ ID NO: 13), 0.5 mM dNTP, 5μl 10×reaction buffer, 1 U pfu DNA polymerase (Stratagene, La Jolla, Calif.),and enough dH₂O to adjust the final volume of the reaction mix to 50 μl.The PCR reaction was initiated with a hot start at 95° C. for 3 minutes,followed by a quick cooling to 4° C. Then, using pfu DNA polymerase(Stratagene, La Jolla, Calif.), 25 thermal cycles of PCR were performedat 94° C. for one minute, 55° C. for 1 minute, and 72° C. for 3.5minutes. The pfu DNA polymerase was used because of its proofreadingactivity and its fidelity. The amplified DNA fragment of 1.3 kb (SEQ IDNO: 17) was separated using agarose gel electrophoresis and subsequentlypurified using Gel Extraction Kit (Qiagen, Valencia, Calif.) accordingto the manufacturer's instructions.

The resulting fragment, synthetic soluble β-secretase (1-1362 bp) (SEQID NO: 17) was inserted into the Topo TA cloning vector (Invitrogen,Carlsbad, Calif.) between the T overhangs. Using the DNA from theresulting construct, expression studies were accomplished by thesubcloning of amino acid residues 14-454 (AGV . . . DEST) and 22-454(TQH . . . DEST) into the BamH1 site of pET11a (Novagen, Madison, Wis.).

Example 2 Refolding and Purification of β-secretase

Synthetic β-secretase (SEQ ID NO: 18) was overexpressed in BL21(DE3)Starcells (Invitrogen, Carlsbad, Calif.). The β-secretase polypeptidesformed inclusion bodies in the cytoplasm of the cells. The cells werelysed and the inclusion bodies were purified by passing the cell lysateover a 27% sucrose cushion. The resulting inclusion bodies weresolubilized at 2 mg/ml in 50 mM CAPS pH 10.7, 8 M urea and 50 mMβ-Mercaptoethanol at room temperature. The solution was then rapidlydiluted 100 fold into rapidly stirring water at room temperature. The pHof the solution was subsequently adjusted to 8.7 and then the solutionwas slowly stirred at room temperature for four hours.

Use of the optimized nucleic acid sequence (SEQ ID NO: 17) resulted inthe expression of BACE polypeptide that is about 4× higher than thewild-type gene.

Subsequently, reshuffling agents were added to the solution. Thestandard condition contained 1 mM reduced glutathione, 0.1 mM oxidizedglutathione, and 1 mM cysteine. However, adjustments in the abovereshuffling ratios improved the efficiency of refolding. For example,the amount of cysteine was fixed while the amounts of reduced andoxidized glutatione was varied. Activity comparisons of refolds, a wayof monitoring the efficiency of refolding, indicated that at day threepost refold, 0.5 mM reduced glutathione:0.5 oxidized glutathioneresulted in an 18× increase in activity while 0.1 mM reducedglutathione:1 mM oxidized glutathione resulted in an 11× increase overthe control condition. Although these conditions resulted in fasterfolding, the final differences with respect to the control at two weekswas 6× and 4×, respectively.

After the reshuffling agents were added, the solution was furtherincubated at room temperature for four hours. The pH of the solution wasthen either maintained at 8.7 or reduced to 4.0 to facilitate refoldingof the protease.

Activity plateaus were reached within three to five days. Upon obtainingmaximal activity, the solution was concentrated one thousand fold andthen subjected to a Superdex 200 gel filtration column (Highload, 26/60,Amersham Pharmacia, Piscataway, N.J.) that was equilibrated with 50 mMTris pH 8.0 with 80 mM urea. The active fractions were pooled and thenloaded onto a Resource Q column (Amersham Pharmacia, Piscataway, N.J.)that was equilibrated with 50 mM Tris pH 8.0 with 80 mM urea. Fractionswere then eluted over 40 column volumes with a final gradientconcentration of 50 mM Tris pH 8.0, 80 mM urea and 500 mM NaCl.

Example 3 Processing of β-secretase

Processing occurred by one of two methods. First, purified β-secretase(SEQ ID NO: 18) was exchanged into 20 mM Hepes pH 7.5 and 150 mM NaCl,and then concentrated to 5 mg/ml and incubated at 4° C. for two weeks.This was the processing method used to generate BACE polypeptide forcrystallization. Alternatively, purified β-secretase (SEQ ID NO: 18) wasexchanged into 20 mM Hepes pH 7.5 and 150 mM NaCl, and then concentratedto 15 mg/ml and incubated at room temperature for 72 hours. Theconcentrations were proposed in order to drive intermolecularinteractions, i.e., to promote a trans cleavage event. The times weredetermined by monitoring processing by SDS-PAGE.

Following trans-cleavage processing (determined by concentrationdependence), which resulted in approximately amino acids 22-45 beingremoved, N-terminal sequencing (equal molar ratios of LRLPRE . . . :LPRE. . . ) and mass spectrometry were conducted to confirm completion ofpropeptide removal and to ensure that no C-terminal truncations tookplace. Trans cleavage processing means proteolysis occurring in anintermolecular fashion, one enzyme “chewing” on a neighboring enzymerather than itself. In this example, it refers to the ability of oneBACE molecule to proteolyze another with a suitable sequence forcleavage, e.g., removal of a propeptide. The equal molar ratios ofLRLPRE . . . (SEQ ID NO: 20):LPRE . . . (SEQ ID NO: 22) showed where thepropeptide is processed, and the ratios indicate the cut is a mixture oftwo species.

Upon completion of processing, the sample was applied to a Superdex 200column (HighLoad, 26/60, Amersham Pharmacia) that was equilibrated in 20mM Hepes pH 7.5 and 150 mM NaCl. The active fractions were then pooledand concentrated to 16 mg/ml for crystallization trials.

Example 4 Enzymatic Activity of Refolded β-secretase

To assess the functionality of β-secretase refolded from E. colioverexpression, a high performance liquid chromatography (HPLC) wasdeveloped using a peptide substrate derived from the sequence of Swedishamyloid precursor protein. The substrate KSEVNLDAEFRK (SEQ ID NO: 16)was used with reverse phase chromatography and was determined to be asuitable substrate for β-secretase with a specificity constant(K_(cat)/K_(m)) of 1800±100 M⁻¹s⁻¹. The substrate (SEQ ID NO: 16) iscleaved between amino acid residues L and D. The activity of thisrefolded β-secretase with this substrate sequence is consistent withβ-secretase derived from other expression systems (Lin, Xinli et al.,“Human aspartic protease mamapsin 2 cleaves the β-secretase site ofβ-amyloid precursor protein”, Proceedings Nat. Acad. Sci., vol. 97, no.4, pp.1456-1460 (2000); Mallender, William M. et al., “Characterizationof Recombinant, Soluble β-Secretase from an Insect Cell ExpressionSystem”, Mol. Pharm., vol. 59, no.3, pp. 619-626 (2001)) and confirmsthat this form of refolded β-secretase is enzymatically active.

Example 5 Crystallization of Refolded Processed β-secretase in thePresence of Inhibitor

The refolded processed BACE was complexed with OM-99-2 (SEQ ID NO: 15),an inhibitor of BACE, at a 1:5 molar ratio. OM-99-2 (SEQ ID NO: 15) waspurchased from Bachem Bioscience Inc. (King of Prussia, Pa.), catalog #H-5108, and is represented by the structure:

It should be noted that OM-99-2 (SEQ ID NO: 15) is a transition statemimetic that is also characterized by the structure EVN{(2R, 4S,5S)-5-amino-4-hydroxy-2,7-dimethyl -octanoyl}AEF.

The complex is then incubated on ice for 5 minutes. The BACE-inhibitorcomplex was screened for crystallization using standard screenspurchased from Hampton Rensearch, Laguna Niguel, Calif., and EmeraldBiostructures, Bainbridge Island, Wash. Crystallization trials werecarried out at 4° C. using the hanging drop method. The drops consistedof 1 μl of reservoir plus 1 μl of the BACE-inhibitor complex. Crystalswere obtained in conditions #12, #37 and #38 of the PEG/ION screen fromHampton Research, Laguna Niguel, Calif. PEG/ION screen condition #12includes 0.2 M ammonium iodide and 20% PEG3350. PEG/ION screen condition#37 includes 0.2 M potassium sodium tartrate tetrahydrate and 20%PEG3350. PEG/ION screen condition #38 includes 0.2 M di-ammoniumtartrate and 20% PEG3350. The crystallization condition from #38 wasoptimized by varying the concentrations of the salt and PEG. Thisyielded the optimal conditions of 20% PEG3350 and 0.2 M di-ammoniumtartrate at 4° C.

Example 6 Crystallization of Apo Refolded Processed β-secretase

Apo BACE was screened for crystallization using standard screenspurchased from Hampton Research and Emerald Biostructures, BainbridgeIsland, Wash. Crystallization trials were carried out at 4° C. using thehanging drop method. The drops consisted of 1 μl of reservoir plus 1 μlof apo BACE. Crystals were obtained in condition #12 of the PEG/IONscreen from Hampton Research, Laguna Niguel, Calif. PEG/ION screencondition #12 includes 0.2 M ammonium iodide and 20% PEG3350. Thecrystallization condition from #12 was optimized by varying theconcentrations of salt and PEG. This yielded the optimal conditions of15% PEG3350 and 0.6 M ammonium iodide at 4° C.

Example 7 Crystallographic Analysis of β-secretase crystallized in thePresence of Inhibitor

The BACE inhibitor complex crystals of Example 5 above were transferredto a solution containing 20% PEG3350, 0.2 M ammonium tartrate and 15%PEG400, and then frozen in liquid propane. Diffraction data wascollected on a Raxis IV detector, purchased from Rigaku/MSC, TheWoodlands, Tex., equipped with osmic focusing mirrors. Two hundredfifteen (215) contiguous 0.5° oscillation images were collected with anexposure time of 6 minutes each. Data reduction with HKL2000 showeddiffraction to 1.7 Å resolution and a 6.5% R-sym. The data were 93%complete with a 4.4 fold multiplicity. The crystals had the space groupof P2₁2₁2₁ with a unit cell of dimensions of a=86.4 Å, b=89.1 Å, andc=131.3 Å. The structure was solved using molecular replacement, asimplemented in CCP4 (Collaborative Computational Project, Number 4,1994, “The CCP4 Suite: Programs for Protein Crystallography”, ActaCryst, D50, pp. 760-763) using 1FKN, a published BACE crystal structurefrom the PDB database, as the search model. There are two molecules inthe asymmetric unit. There is clear density for the protein. Refinementwas carried out with CNX (Accelrys, San Diego, Calif.) and yielded afinal R of 0.18 and a R_(free) of 0.21.

Example 8 Crystallographic Analysis of Uncomplexed β-secretase

The uncomplexed BACE crystals of Example 6 above were transferred to asolution containing 20% PEG3350, 0.6 M ammonium iodide and 15% glycerol,and then frozen in liquid propane. Diffraction data were collected atthe Industrial Macromolecular Crystallography Association, Argonne,Ill., beamline located at the Advanced Photon Source. Diffraction datawas collected on a Q210 detector, purchased from ADSC, Poway, Calif.Four hundred (400) contiguous 0.5° oscillation images were collectedwith an exposure time of 2 seconds each. Data reduction with HKL2000,HKL Research, Inc., Charlottesville, Va., showed diffraction to 2.2 Åresolution and a 6.5% R-sym. The data were 99.8% complete with a 4.1fold multiplicity. The crystals had the space group of C2 with a unitcell of dimensions of a=236.0 Å, b=103.6 Å, and c=65.0 Å. The structurewas solved using molecular replacement, as implemented in CCP4(Collaborative Computational Project, Number 4, 1994, “The CCP4 Suite:Programs for Protein Crystallography”, Acta Cryst., D50, 760-763) using1FKN, a published BACE crystal structure from the Protein Data Bankdatabase as the search model. There are three molecules in theasymmetric unit. There is clear density for the protein. Refinement withCNX (Accelrys, San Diego, Calif.) yielded an R factor of 25.5% and anR_(free) of 29.7%. LENGTHY TABLE REFERENCED HEREUS20070099273A1-20070503-T00001 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070099273A1-20070503-T00002 Please refer to the end of thespecification for access instructions. LENGTHY TABLE The patentapplication contains a lengthy table section. A copy of the table isavailable in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070099273A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated or recombinant nucleic acid comprising the nucleotidesequence set forth in SEQ ID NO:
 1. 2. The isolated or recombinantnucleic acid of claim 1 wherein said nucleic acid comprises thenucleotide sequence set forth in SEQ ID NO:
 17. 3. An expression vectorcomprising the nucleic acid of claim
 2. 4. A host cell comprising thevector of claim
 3. 5. A method for making β-secretase polypeptidecomprising transforming a host cell with the vector of claim 3 underconditions in which said polypeptide is expressed.
 6. The method ofclaim 5 further comprising a refolding step wherein said polypeptide isrefolded in the presence of about 0.5 mM reduced glutathione and about0.5 mM oxidized glutathione.
 7. The method of claim 5 further comprisinga processing step wherein said polypeptide is exchanged into about 20 mMHepes at about pH 7.5 and about 150 mM NaCl, and then concentrated toabout 5 mg/ml and incubated at about 4° C. for about two weeks to form aprocessed polypeptide.
 8. The method of claim 7 wherein said processedpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
 9. The method of claim 7 wherein said processed polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:
 22. 10. Amethod for growing a crystal comprising: (a) adding about 16 mg/ml of apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:22 to a crystallization solution, said solution comprising about 13.75%to about 15% PEG3350 and about 0.6 M ammonium iodide; and (b)crystallizing said solution at about 4° C. using a hanging drop method.11. A method for growing a crystal comprising a polypeptide complexed toa ligand comprising: (a) adding about 16 mg/ml of a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 22 and about0.5 mM to about 1.0 mM of said ligand to a crystallization solution,said solution comprising about 20% PEG3350 and about 0.2 M ammoniumtartrate; and (b) crystallizing said solution at about 4° C. using ahanging drop method.
 12. The method of claim 11 wherein said ligand isan antagonist.
 13. A crystal made by the method of claim
 10. 14. Acrystal of an uncomplexed β-secretase polypeptide wherein saidβ-secretase polypeptide is expressed in E. coli cells comprising apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:22, wherein said crystal effectively diffracts X-rays for determinationof atomic coordinates of said polypeptide to a resolution of greaterthan about 5.0 Å.
 15. A magnetic data storage medium comprising thestructure coordinates set forth in Table
 1. 16. A computer for producinga three-dimensional representation of β-secretase polypeptide which isdefined by the structure coordinates set forth in Table 1, or athree-dimensional representation of a homologue of said β-secretasepolypeptide wherein said homologue has a root mean square deviation fromthe backbone atoms set forth in Table 1 of less than about 1.5 Å,wherein said computer comprises: (a) a machine-readable data storagemedium comprising a data storage material encoded with machine-readabledata, wherein said data comprises the structure coordinates set forth inTable 1; (b) a working memory for storing instructions for processingsaid machine-readable data; (c) a central-processing unit coupled tosaid working memory and to said machine-readable data storage medium forprocessing said machine readable data into said three-dimensionalrepresentation; and (d) a display coupled to said central-processingunit for displaying said three-dimensional representation.
 17. A methodfor identifying a ligand that binds to β-secretase comprising: (a)obtaining a set of atomic coordinates defining the three-dimensionalstructure of a crystal of an uncomplexed, processed β-secretasepolypeptide expressed in E. coli cells that effectively diffracts X-raysfor determination of the atomic coordinates of said β-secretasepolypeptide to a resolution of greater than about 5.0 Å; (b) selecting aligand by performing rational drug design with said set of atomiccoordinates obtained in step (a), wherein said selecting is performed inconjunction with computer modeling; (c) contacting said ligand with saidpolypeptide; and (d) detecting binding of said ligand to saidpolypeptide.
 18. A method for identifying a β-secretase antagonistcomprising the steps of: (a) selecting a potential antagonist byperforming rational drug design using the three-dimensional structure ofa crystal according to claim 14, wherein said selecting is performed inconjunction with computer modeling; (b) contacting said potentialantagonist with β-secretase; and (c) detecting binding of said potentialantagonist to said β-secretase, wherein an antagonist is identified onthe basis of its ability to inhibit the catalytic activity of saidβ-secretase.
 19. A method for identifying an inhibitor of β-secretasecomprising: (a) obtaining a set of atomic coordinates from a crystaldefining the three-dimensional structure of an uncomplexed, processedβ-secretase polypeptide expressed in E. coli cells; (b) selecting apotential inhibitor by performing rational drug design with said set ofatomic coordinates obtained in step (a), wherein said selecting isperformed in conjunction with computer modeling; (c) contacting saidpotential inhibitor with a β-secretase protein; and (d) measuring theactivity of said protein, wherein said potential inhibitor is identifiedwhen there is a decrease in activity of said β-secretase in the presenceof said inhibitor as compared to the activity of β-secretase in theabsence of said potential inhibitor.
 20. A method for identifying apotential inhibitor of β-secretase comprising the steps of: (a) viewinga three-dimensional structure of said β-secretase as defined by theatomic coordinates of β-secretase set forth in Table 1; (b) employingsaid three-dimensional structure to design or select said potentialinhibitor; (c) synthesizing said potential inhibitor; and (d) contactingsaid potential inhibitor with said β-secretase in the presence of asubstrate to determine the ability of said potential inhibitor toinhibit said β-secretase.